The goal of these studies is to understand the regulatory interactions of key signaling proteins targeted to plasmalemmal caveolae in vascular endothelial cells, with an ultimate aim of identifying the abnormalities in endothelial signaling that underlie many vascular disease states. Caveolae serve as sites for the sequestratior of signaling molecules, including heterotrimeric G proteins, G protein-coupled receptors, the calcium regulatory protein calmodulin, the endothelial isoform of nitric oxide synthase (eNOS), thc. scaffolding/regulatory protein caveolin, and diverse protein kinases and lipid kinases and their substrates. Caveolin serves both a structural and regulatory role in caveo!ae. These studies will explore the role of caveolin in modulating three important protein-protein interactions in caveolae: the associations of G protein subunits with one another; the interaction of G protein betagamma (Gbetagamma) subunits with the B isoform of phosphoinositide 3-kinase B (P13-Kbeta); and the activation by calmodulin of eNOS. These studies will identify the subunits and domains of P13-Kbeta that interact with Gvetagamma. We will explore the role of caveolin in the regulation of phosphoinositide targeting and metabolism in caveolae. We will extend our studies of signaling protein interactions using cell imaging approaches, applying fluorescence resonance energy transfer (FRET) methods to understand the spatial and temporal relationships of receptor-regulated Galpha-Gbetagamma associations, the interaction of calmodulin and eNOS, and the regulation by caveolin of calcium dynamics ii living endothelial cells. We have discovered that calmodulin in endothelial cells is phosphorylated, and tha calmodulin phosphorylation modifies its ability to activate eNOS. We will construct and characterize calmodulin phosphorylation mutants, and will use both biochemical as well as FRET-based approaches to understand the role of calmodulin phosphorylation in endothelial signaling. These studies will combine studies in purified systems with analyses in intact cells, and may lead to the identification of new sites fo pharmacological regulation, and provide insight into the mechanisms that underlie endothelial dysfunction.